The biochemical basis for the regulation of hormone-responsive adenylate cyclase of rat hepatocytes by soluble cytosolic components will be investigated. High-speed supernatants of whole homogenates can restore catecholamine-sensitive adenylate cyclase activity of purified hepatocyte membranes, reconstitute guanine mucleotide- and fluoride sensitive adenylate cyclase activity of S49 mouse lymphoma cyc- membranes, and enhance the action of cholera toxin on hepatocyte cyclase activity and the toxin-catalyzed AD32p-ribosylation of hepatocyte membrane proteins. Using each of the above mentioned phenomena as a means to assay, identify, and follow these activities of the soluble factors, we plan to characterize the physical and chemical properties of molecules responsible for these activities. We will ascertain if these components are naturally soluble components, and if these activities reside in a single or in several discrete molecules. The properties of these soluble regulators will be defined by gel filtration and affinity chromatography, polyacrylamide gel electrophoresis, sedimentation velocity analysis, and will be compared to those of the membrane-bound adenylate cyclase regulatory proteins previously identified by others. The nature of these soluble components which modulate several key facets of adenylate cyclases responsiveness to the action of hormones and other agents will be investigated in detail. Thyroid status modulates catecholamine-sensitivity of fat cell adenylate cyclase without affecting hormone receptor number or the amount of catalytic cyclase. To define the basis for this "permissive effect" we plan to study the functional and physical status of the cyclase regulatory proteins of fat cell membranes from euthyroid, hypothyroid, and hyperthyroid rats. Functional capacity will be assayed by reconstitution studies (with S49 cyc- recipient membranes), and the physical and chemical nature of putative regulatory proteins will be defined after toxin-catalyzed AD32p-ribosylation as described above. In addition, peptide mapping and 2-dimensional gel analysis of the toxin targets will be performed. By employing these complementary strategies we seek to identify and characterize these physiologically important modulators of the response of adenylate cyclases to hormonal stimulation.